Herpes simplex virus (HSV), and likely all herpesviruses, use two methods to enter cells: (1) direct fusion: the virus fuses its lipid bilayer with the plasma membrane at the cell surface and (2) receptor-mediated endocytosis (RME): the virus fuses with an endosomal membrane after endocytosis and a reduction in pH. Our long-term goal is to understand the molecular events occurring during HSV-1-induced membrane fusion for both direct fusion and RME. We have recently identified a fusion intermediate (hemifusion) during virus-to- cell fusion for HSV-1. This is the first description of a fusion intermediate for any herpesvirus or any virus that uses more than two proteins to mediate membrane fusion. Our preliminary data are consistent with hemifusion occurring at the cell surface in cells that require endocytosis and low pH for HSV-1 entry. The focus of this application is to examine membrane fusion in our newly proposed model for herpesvirus RME. A unique aspect of this model is the proposed cell-surface location of the hemifusion event. We outline experiments here to establish the cellular location of hemifusion during HSV-1 RME by co- localizing the site of hemifusion with cellular markers. We will also use pharmacological agents that block endocytosis and endosomal pH reduction to determine if those processes are required for hemifusion to occur. Because herpesvirus entry occurs at the cell surface at neutral pH and is postulated to occur at the low pH of a late endosome, a testable prediction is that the HSV-1 fusion machinery must function at both neutral and low pH. To test that prediction, we will examine HSV-1 membrane fusion at low pH and compare it to that at neutral pH. The results obtained from this application will establish a model for HSV-1 RME. A detailed understanding of membrane fusion induced during direct fusion and RME is necessary to facilitate the design of inhibitors to block hemifusion, full fusion, and virus spread. PUBLIC HEALTH RELEVANCE: The aim of this project is to characterize the membrane fusion events during herpes simplex virus type 1 (HSV-1) entry into cells via receptor-mediated endocytosis. HSV-1 enters cells by multiple routes so the intricate understanding of the mechanism of each route is required to facilitate the design of anti-virals to block virus entry and spread.